21-hydroxylation of cyclopentanophenanthrene derivatives by aspergillus niger



United States Patent 2,812,285 Patented Nov. 5, 1957 lice . NANTHRENE DERIVATIVES BY ASPERGILLUS NIGER Alejandro Zalfaroni and Benjamin A. Rubin, Mexico City,

Mexico, assignors to Syntex, S. A., Mexico City, Mexico, a corporation of Mexico No Drawing. ApplicationApril 23, 1954, Serial No. 425,302

2 Claims priority, application Mexico April 30, 1953 12 Claims. (Cl. 195-51) The present invention relates to a novel method for the preparation of cyclopentanophenanthrene derivatives. More particularly the present invention relates to the oxidative introduction of a hydroxyl group at position 21 of steroidal compounds of the pregnane and 19-nor pregnane series by the action of the fungus Aspergillus nzger.

In the United States Patent Serial No. 2,602,769, of Murray and Peterson, issued July 8, 1952, there is disclosed the introduction of oxygen into the steroid molecule by means of Mucorales fungi. Although the aforementioned patent refers generally to the introduction of oxygen into the steroid molecule by various members of the Mucorales order, it would appear from this patent that the action of the variousfungi of this order is selective with regard to specific steroids and with regards to positions or position oxygenated. In general, introduction of oxygen at positions C-6, C-7, C-8, C-11 and C-14 of the steroidal molecule is disclosed. In United States Patent Serial No. 2,649,402, issued August 18, 1953, the same inventors disclose the introduction of oxygen into the steriod molecule by fungi of the genus Aspergillus and especially Aspergillus niger. Here again the action of the microorganism insofar as it is clearly described appears to be specific, and only the introduction of oxygen into position C-1l of the steroid molecule is disclosed in this patent. Although this patent refers to varying times of contact between the steroids and the fungus, it is pointed out that 24 hours is generally satisfactory and in the specific examples this period of time is generally used with some few examples referring to an incubation period or bioconversion time of 48 hours.

In accordance with the present invention, it has been discovered that when a steroid is incubated with the microorganism Aspergillus niger and especially a specific strain thereof, namely ATCC 9142, for periods of time in excess of 48 hours and preferably for a period of time between 96 to 120 hours, a hydroxy group is introduced in position 21 of a steroid compound of the pregnane or 19-nor pregnane series.

The present invention therefore provides a method for the preparation of important steroidal hormones such as desoxycorticosterone, corticosterone and other similar compounds starting from the corresponding 21- desoxy compounds. In general the method of the present invention has been found applicable to substantially all 21-desoxy steroids of the pregnane series, including those compounds having a 3-keto group or double bond, as for example between C-4 and C-5, or in other positions on the steroidmolecule as well as hydroxy groups or keto groups in position 11 or a hydroxy group in position 17 of the steroid molecule. The pregnane compounds may be saturated and in this case the compounds may be the C- normal compounds or the C-5 allo compounds. Similar pregnane compounds of the 19-n0r series may also be utilized, 1. e. those compounds having the angular methyl group in this position replaced with hydrogen.

In practicing the process of the present invention, various conditions well known in the art for promoting the active growth of the fungus may be utilized, as for example those conditions specifically set forth in the Murray and Peterson Patent No. 2,602,769. Thus in general a suitable nutrient medium is provided, inoculated with the fungus and thereafter kept under aerobic conditions in order to promote active growth of the fungus. Thereafter the steroid to be oxygenated is added, preferably in solution in a suitable solvent such as ethyl alcohol, dispersed in the medium by agitation and the incubation is prolonged for a substantial period under the same conditions which allowed profuse growth of the fungus. The reaction medium is then extracted with a suitable solvent for the steroid such as dichloroethylene. The extracts are then evaporated to dryness and the residue is purified as by solution in a solvent and chromatography on a suitable column. Unlike the conditions, however, in the aforementioned Murray and Peterson patents, the incubation should be prolonged for a sufiicient length of time in order to produce an economic yield of the 2l-hydroxy product. It has been found, for example, that the period of incubation of 24 hours produces no detectible amount of 21-hydroxy compound. It is therefore desirable that a period of time be utilized in excess of 48 hours, and in most cases for optimum results a period of time of between approximately 96 to 120 hours should be utilized, although. in some instances best results have been obtained with incubation of a week or more. i

The following specific procedure was found to be satisfactory for the oxygenation and separation of the oxygenated steroids.

A liquid medium was prepared containing per 1000 cc., 20 g. of peptone and 50 g. of Karo syrup in sterile tap water. The medium was then divided in 200 cc. portions and each portion was poured into a 1000 cc. Erlenmeyer flask. After sterilizing, the contents of each flask was inoculated with a suspension of spores of Aspergillus niger (ATCC 9142). The mixture was incubated at 28 C. for 24 to 48 hours with rotatory agitation. In the same way, 30 liters of the liquid medium was prepared in a 50 liter Pyrex bottle and then the contents of S of the previously mentioned Erlenmeyer flasks (totalvolume of liquid 1000 cc.) was added to the liquid medium. 10 g. of the steroid to be oxidized were then dissolved in 100-200 cc. of ethyl alcohol and the alcoholic solution was added to the bottle containing the reaction medium and the mixture was then homogenized. This mixture was then incubated for 96- hours at 28 C. While stirring with a stainless steel mechanical stirrer at 174 R. P. M., the stirrer being equipped with metallic attachment to introduce and disperse sterile air at the rate of 100 liters per minute.

After the incubation period, the steroids were extracted. For this purpose the reaction medium was extracted with 5 successive fractions of 10 liters each of ethylene dichloride. After evaporation to dryness of the combined extracts a residue remained weighing between 15 and 20 g. This residue was dissolved in benzene and chromatographed in a column containing 1 kilogram of silica. The first fraction eluted from the column by routine methods utilizing successively more polar solvents, always consisted of unchanged steroidal starting material and the second and/or successive fractions consisted of oxygenated steroids. In each instance the oxygenated steroids were separated and purified by conventional methods such as crystallization, etc.

Example vII Starting from 10 -g..of lla-hydroxyprogesterone (A pregnen-l1a-ol-3,20-dione) there was obtained 6.9 g. of steroids, of which 2.9 -g. proved to be epi-corticosterone (M-pregnene-l1a,2l-diol-3,20-dione) having a melting point of 147-150 C.

Example Il'I Starting from 10 g. of llfi-hydroxyprogesterone (A I pregnen-l1,8-01-3,20-dione) there was obtained 6.95 g. of steroids, of which 1.95 'g. proved to be corticosterone (M-pregnene-l1,6,21-diol-3,20-dione) having a melting point of 179-182 0.

Example I V Starting from 3 g. of l-l-keto-progesterone (A -pregmen-3,11,20-trione), the preparation of which has been described, for example, by Reichstein, Helvetica Chimica Acta, 23, 684 (1940), there was obtained 2.4 g. of steroids, of which 1.2 g. proved to be Kendalls compound A (11- dehydrocorticosterone; A -pregnen-21-ol-3,11,20- trione), having a melting point of 178179 C.

Example V Starting from 10 g. of 17a-hydroxyprogesterone, 7.0 g. of steroids were recovered, of which 1.8 g. was Reichsteins Substance S (A -pregnene-17a,21-diol-3,20-dione) having .a melting point of 204.207 C.

Example VI Starting from 5 g. of 6,8-hydroxyprogesterone, 3.4 g. of steroids were recovered, of which 1.3 g. was A -pregnene-6fl,21-diol-3,20 dione (65 hydroxydesoxycorticosterone) having a melting point of 198-200 C.

Example VII Starting from 10 g. of nor-progesterone, prepared according to United States application Serial No. 250,036,

filed October 5, 1951, 8.1 g. of steroids were recovered,

4.8 g. of which proved to be A -19-nor-pregnen-21-ol- 3,20-dione (nor-desoxycorticosterone).

Acetylation of this compound with acetic anhydride in pyridine solution (1 hour at the temperature of the steam bath) afforded the acetate of A -19-nor-pregnen- 2l-ol-3,20-dione having a melting point of 168170 C.

We claim:

1. A process for the production of a steroidal 21-hydroxy compound selected from the group consisting of compounds of the pregnane series and 1*9-norpregna1i5 series which comprises subjecting a corresponding 21- desoxy compound to the oxidizing action of a strain of Aspergillus niger by dispersing said 21- desoxy compound in a nutrient medium containing the fungus in active growth condition to form a reaction mixture, maintaining said mixture under conditions favorable tofungus growth for a period of time exceeding 48 hours and recovering the formed ZI-hydroxy compound from said mixture.

2. The process of claim 1 wherein the fungus is Aspergillas n'iger ATCC 9142 and the 2l-desoxy compound is subjected to the action thereof for a period of time between approximately :96 and 120 hours.

3. The process of claim 1 wherein the 21-hydroxy compound is a. A -3-ketone of the pregnane series.

4. The process of claim 1 wherein the Z-hydroxy com pound is a A -3-ketone of the 19-nor-pregnane series.

5. The process of claim 1 wherein the 21-hydroxy compound is a member of the pregnane series.

.6. The process of claim 1 wherein the 21-hydroxy compound is a member of the 19-nor pregnane series.

7. The process .of claim 1 wherein the product is desoxycorticosterone .and the starting compound is progesterone.

8. The process of claim 1 wherein the product is epicorticosterone .and the starting compound is lla-hydroxyprogesterone.

.9. The process of .claim 1 wherein the product is cor-- ticosterone and the starting compound is ,ILB-hydroxyprogesterone.

10. The .process of claim 1 wherein the product is A -pregnen-l7a,2l-diol-3,20-.dione .and the starting compound is A -pregnen-17a-ol-3,20-dione.

11. The process of claim 1 wherein the product is A -pregnen-6fl,21-diol-3.,20-dione and the starting compound is A -pregnen-6/3-ol-3,20-dione.

12. The process of claim 1 wherein the product is A -19-nor-pregnen-21-ol-3.,20-dione and the starting compound is A -19-nor pregnen-3,20dione.

References Cited in the file of this patent UNITED STATES PATENTS 2,649,402 Murray Aug. 18, 1953 

1. A PROCESS FOR THE PRODUCTION OF A STEROIDAL 21-HYDROXY COMPOUND SELECTED FROM THE GROUP CONSISTING OF COMPOUNDS OF THE PREGNANE SERIES AND 19-NOR PREGNANE SERIES WHICH COMPRIES SUBJECTING A CORRESPONDING 21DESOXY COMPOUND TO THE OXIDIZING ACTION OF A STRAIN OF ASPERGILLUS NIGER BY DISPERSING SAID 21-DESOXY COMPOUND IN A NUTRIENT MEDIUM CONTAINING THE FUNGUS IN ACTIVE GROWTH CONDITION TO FORM A REACTION MIXTURE, MAINTAINING SAID MIXTURE UNDER CONDITIONS FAVORABLE TO FUNGUS GROWTH FOR A PERIOD OF TIME EXCEEDING 48 HOURS AND RECOVERING THE FORMED 21-HYDROXY COMPOUND FROM SAID MIXTURE. 